(CANCER RESEARCH 50. 48-53, January I, 1990]
Insulin-like Growth Factor Receptor Expression and Function in Human Breast
Cancer1
Kevin J. (alien, Douglas Yee, William S. Sly, James Perdue, Brian Hampton, Marc E. Lippman, and Neal Rosen
Vincent T. Lombardi Cancer Research Center, Georgetown University, Washington, DC 20007 [K. J. C., D. Y., M. 'E. L.. N. R.J; St. Louis University School of Medicine,
St. Louis, Missouri 63104 ¡W.S. S.J; and the Holland Research Labs, American Red Cross, Rockville, Maryland 20850 [J. P., B. H.J
both IGF-I and IGF-II may have a growth-promoting role in
human breast cancer.
Distinct receptors for IGF-I and IGF-II have been described
and cloned. The type I IGF receptor (IGF-I receptor) is a
transmembrane heterotetramer structurally very similar to the
insulin receptor. It is composed of two «chains which comprise
the extracellular ligand binding domain, and two ,i chains which
include the transmembrane region as well as an intracellular
tyrosine kinase domain (9, 10). In contrast, the type II IGF
receptor (IGF-II receptor) exists as a single transmembrane
chain with a small intracellular domain lacking apparent tyrosine kinase activity (11). The type II IGF receptor is also the
receptor for marinóse 6-phosphate (12), a sugar moiety found
in several glycoproteins which is important in the recognition
and intracellular transport of soluble lysosomal enzyme precur
sors. Additionally, the type II receptor binds procathepsin D, a
proteolytic enzyme produced in abundance by some breast
cancer cells, which has also been reported to be a mitogen for
breast tumor epithelial cells in culture (13).
Considerable cross-reactivity takes place between the various
ligands and receptors in the insulin/insulin-like growth factor
family (14). Insulin can bind to both the insulin receptor and
the type I IGF receptor, while IGF-I and IGF-II can bind to
the insulin receptor as well as the type I and type II IGF
receptors. Evidence from a number of previous studies has
suggested that at least some of the biological effects of IGF-II
are mediated by the type I IGF receptor (15, 16). In human
breast cancer, inhibition of growth of some tumor cell lines can
be accomplished in vivo (17) or in vitro (18) by an antibody
which blocks ligand binding to the type I IGF receptor.
In this study we characterize the expression of the type I and
type II IGF receptors in human breast cancer by examining
mRNA expression and through ligand binding studies. Addi
tionally, through the use of antibodies against the type I and
type II IGF receptors, we explore the biological role of the two
receptors in a model breast cancer cell line, MCF-7. The exper
iments suggest that both type I and type II IGF receptors are
ubiquitous in human breast cancer but that the mitogenic effects
of both IGF-I and IGF-II are mediated by the type I receptor.
ABSTRACT
The insulin-like growth factors IGF-I and Idi -li are potent mitogens
for several breast tumor cell lines in culture. Additionally, both IGF-I
and IGF-II mRNAs are easily detected in the majority of breast tumor
specimens examined, while no breast cancer epithelial cell lines we have
studied express authentic IGF-I mRNA, and few lines express IGF-II
iiiRNA. Although receptors for insulin, IGF-I, and IGF-II have been
described, there is significant cross-reactivity between the various recep
tors and ligands in the insulin/insulin-like growth factor family, and it is
not clear which receptor or receptors are responsible for the biological
effects of these growth factors in this system.
Using an RNase protection assay, we examined breast tumor speci
mens and breast cancer epithelial cell lines for expression of mRNA
encoding the type I and type II IGF receptors as well as the insulin
receptor. Virtually all of the specimens examined expressed mRNA for
all three receptors. We then examined estrogen-dependent MCF-7 cells
for the mitogenic effects of IGF-I and II in the presence of antibodies to
both the type I and type II receptors. aIR-3, a monoclonal antibody
which blocks the type I receptor, abolished the mitogenic effects of both
IGF-I and IGF-II. It did not, however, block the mitogenic effects of
insulin. We conclude that type I and type II IGF receptors are ubiqui
tously expressed in breast cancer, and our experiments with MCF-7 cells
suggest the mitogenic effects of both IGF-I and IGF-II are mediated via
the type I IGF receptor.
INTRODUCTION
In recent years, an extensive literature has developed on the
role of peptide growth factors in the growth regulation of human
breast cancer and other malignancies (1). These factors may act
by endocrine, autocrine, or paracrine pathways to regulate
tumor growth. Among the growing list of peptides which may
have biological importance in this system are the insulin-like
growth factors IGF-I and IGF-II.1 As their name implies these
proteins are closely related to insulin, or more accurately, proinsulin (2-4). IGF-I is also known as somatomedin C and is
the mediator of the effects of growth hormone, and so it
functions as an important regulator of normal growth and
development. Insulin-like growth factor II, on the other hand,
has been postulated to be a growth regulator during fetal
development but has no defined function in the adult. In rodent
model systems, IGF-II production appears to decline at birth,
while in humans, measurable quantities of IGF-II continue to
be produced throughout adult life (5).
Both IGF-I and IGF-II have been shown to be potent mito
gens for a number of breast cancer epithelial cell lines in vitro
(6, 7) and mRNA for both IGF-I and IGF-II is detectable in
the majority of human breast tumors (6, 8). This suggests that
MATERIALS
AND METHODS
Cells and Cell Culture
MCF-7 cells were originally obtained from Dr. Marvin Rich of the
Michigan Cancer Foundation. Hs578T cells were a gift of Dr. Adeline
Hackett of the Peralta Cancer Institue, Oakland, CA. ZR75-B-cells
were subcloned from ZR7S-1 cells, a line initially established at the
National Cancer Institute (19). All other cell lines were obtained from
the American Type Culture Collection, Rockville, MD. All cell lines
were maintained in phenol-red free IMEM (Biofluids, Rockville, MD)
supplemented with 10% fetal calf serum (GIBCO, Detroit, MI).
Received 6/29/89; revised 9/27/89; accepted 10/3/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported in part by a grant from the Ladies Auxiliary to the Veterans of
Foreign Wars.
3The abbreviations used are: IGF-I, insulin-like growth factor I; IGF-II,
insulin-like growth factor II; IMI-AI, improved minimum essential medium; PBS,
phosphate buffered saline; cDNA, complementary DNA; SDS, sodium dodecyl
sulfate.
Growth Studies
MCF-7 cells were washed three times for 5 min in PBS at 37°C.The
cells were then briefly trypsinized, and plated in 24-well clusters at a
density of 25,000 cells per well in 0.5 ml phenol-red free IMEM
48
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1990 American Association for Cancer Research.
IGF RECEPTORS
IN BREAST
CANCER
fragment including bases 2958-3335, which was ligated into a pGem 4
vector (Promega). This template was used to synthesize antisense RNA
probes for both RNase protection assay and northern blot analysis.
Insulin Receptor. cDNA coding for the .; chain of the human insulin
receptor was provided in a Bluescript vector (Stratagene, La Jolla, CA)
by Dr. David Lebwohl. This was linearized with Stul providing a
template for a 600-base pair complementary riboprobe comprising the
carboxy terminal end of the ßchain and a small portion of the 3'-
containing 5% calf serum which had been stripped of estrogen by
sulfatase and dextran-coated charcoal treatment (20). After 24 h. the
medium was removed and the wells were refilled with 0.5 ml serumfree medium consisting of phenol red-free IMEM with 2 mg/liter
transferrin, 2 mg/liter fibronectin, 20 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer, 292 mg/liter glutamine, and 1x trace
elements (Biofluids). After an additional 24 h, the serum-free medium
was replaced, and growth factors and antibodies were added at the
concentrations shown. For growth studies involving antibodies, cells
were incubated with antibody alone for 1 h before the competing growth
factor was added. Media and growth factors were replaced after 48 h
of treatment, and at 96 h the cells were harvested in PBS with 0.02%
EDTA and counted on a hemocytometer. All values represent counts
from duplicate wells ±standard error.
Recombinant human IGF-I was obtained from Amgen, Inc., (Thou
sand Oaks, CA). IGF-II was isolated from pooled human Cohn fraction
IV, as previously described (21). «IR-3was the gift of Dr. Steven
Jacobs. Recombinant human insulin (Humulin R) was purchased from
Eli Lilly (Indianapolis, IN). Control mouse and rabbit IgGs were
obtained from Sigma (St. Louis, MO). Antibodies against the type II
IGF receptor have been previously characterized (22).
untranslated end of the receptor.
Northern Blot Analysis
Ten /ig of total RNA from breast cancer cell lines were electropho
resed on a 1.1% agarose gel containing 7.2% formaldehyde. The RNA
was transferred by capillary action to a nylon membrane (Nytran,
Schleicher, and Schuell, Keene, NH). Riboprobe for the type II IGF
receptor was prepared from the cDNA template described above ac
cording to the manufacturer's protocol (Promega). The blots were
hybridized in 50% formamide, 450 mM NaCl, 45 m\i sodium citrate,
100 Mg/ml tRNA, and 10% dextran sulfate overnight at 42'C. Blots
were washed at room temperature with four changes 300 mM NaCl, 30
mM sodium citrate, 0.1 % SDS for a total of 1 h, followed by a 15-min
wash in 15 HIMNaCl, 1.5 mM sodium citrate, 0.1% SDS at 65°C.Blots
were exposed to X-ray film using an intensifying screen for 1-4 days at
-70°C.
IGF Binding Studies
MCF-7 cells were plated in six-well plates in phenol red-free IMEM
containing 5% charcoal stripped serum at a density of 200,000 cells/
well. When the cells reached confluence, they were switched overnight
to serum-free medium. Immediately before the binding study, the cells
were washed three times with PBS at 37°Cand then 2 ml binding buffer
(0.1 M 4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid, 0.12 M
NaCl, 0.005 M KCI, 0.0012 M MgSO4, 0.008 M glucose) was added to
each well at 4'C. For IGF-I binding studies, 30,000 cpm '"I-IGF-I
RNase Protection Assay
Riboprobes were synthesized from the cDNA templates described
above according to the manufacturer's protocol (Promega). Thirty pg
of total RNA for each sample was hybridized overnight at 50"C with
50,000 cpm probe in 30 n\ buffer containing 80% formamide, 40 mM
piperazine-/VJV'-bis(2-ethanesulfonic acid), 0.4 M NaCl, 0.1 M EDTA.
(specific activity, 2000 Ci/mmol; Amersham, Arlington Heights, IL)
were added to each well along with competing ligand or antibody. For
IGF-II binding studies, human IGF-II from Cohn fraction IV, was
radioiodinated by the lactoperoxidase method to a specific activity of
200 Ci/mmol. 50,000 cpm was added to each well along with competing
ligand or antibody. Binding with both IGF-I and IGF-II was carried
out for 4 h at 4"C. Antibody competition studies were performed
following a 1-h antibody preincubation at 4°C.After 4 h, cell mono-
Samples were subsequently digested with 40 ¿ig/mlRNase A (Sigma)
for 30 min at 25"C. Digestion was terminated with proteinase K and
SDS. The samples were extracted with phenol:chloroform:isoamyl al
cohol (20:20:1) and then were precipitated on dry ice with 1 >m of
tRNA (Sigma) and two volumes of absolute ethanol. The pellets were
resuspended in 5 p\ of an 80% formamide loading buffer, and run on a
6% polyacrylamide sequencing gel with 8 M urea. Size markers were
prepared by end labeling A/spI-digested fragments of pBR322 (New
England Biolabs, Beverly, MA). The gels were dried and exposed to Xray film in the presence of an intensifying screen at -70°C for I to 3
layers were washed briefly with iced PBS and harvested in 0.5 M NaOH.
Bound IGF was determined on a Packard gamma counter.
days.
RNA Studies
Nucleic Acid Preparation
RESULTS
Patient tumor specimens were obtained in the operating room. A
portion of the tissue was sent for routine histopathology and the
remainder was frozen on dry ice, and subsequently stored at -70°C.
RNA Studies
Total RNA from breast cancer cell lines and from patient tumor
specimens was extracted using the guanidine/cesium chloride method
(23). Cells were harvested from subconfluent tissue culture flasks di
rectly in guanidinium solution. Frozen tumor tissue was pulverized on
dry ice, and the resulting powder was suspended in guanidinium solu
tion and then homogenized with a Polytron apparatus (Brinkmann,
Westbury, NY). After ultracentrifugation on a cesium cushion, the
quantity of RNA was determined spectrophotometrically, and aliquots
were electrophoresed on 1.1% agarose minigels containing 7.2% form
aldehyde and stained with ethidium bromide to confirm RNA integrity.
Type I IGF Receptor. RNase protection assay of total RNA
from breast cancer cell lines was positive for type I IGF receptor
message in all nine specimens tested (Fig. 1). This included
estrogen receptor-positive breast tumor cell lines (MCF-7, T47D, ZR75-B, CAMA-1) as well as estrogen receptor-negative
tumor cell lines (MDA-MB 231, HS578-T, MDA-MB 468,
MDA-MB 436, BT-549). HBL-IOO represents a cell line de
rived from breast milk, and 172 is a diploid cell line derived
cDNA Probes
Type I IGF Receptor. cDNA for the ß
chain and a portion of the 3'-
•¿
r-^-rnr
untranslated region of the type I IGF receptor was provided by Dr.
Axel Ullrich (24). The region including bases 2737-3030 was spliced
into a pGem-3 vector (Promega, Madison, WI) and subsequently li
nearized with EcoRl. This provided a template coding for a 299-base
pair complementary probe which includes the transmembrane portion
of the 0 chain.
Type II IGF Receptor. The 9-kilobase cDNA for the type II IGF
receptor (25) was digested with BamHl producing a 377-base pair
f
«,
-404
.
Fig. I . RNase protection assay for type I IGF receptor mRNA in breast cancer
cell lines and tumor specimens. A 293-base pair protected fragment is seen in all
cell lines. The patient tumor specimens represented by individual initials (A. W.,
L. C., W. J., S. J.) also express detectable mRNA. Radiograph exposed for 48 h.
49
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1990 American Association for Cancer Research.
IGF RECEPTORS
IN BREAST
from a reduction mammoplasty. Of a total of seven patient
breast tumor specimens examined, six contained detectable type
I IGF receptor message (Fig. 1 and data not shown).
Type II IGF Receptor. All 11 breast cancer cell lines examined
expressed mRNA for the type II IGF receptor by northern
analysis (Fig. 2). A single 9.4-kilobase transcript was seen,
corresponding to the message size reported in the literature
(25). By RNAse protection assay, eight of eight breast tumor
cell lines expressed mRNA for the type II IGF receptor (Fig.
3). In the same figure, six of six cell lines from other tumors
expressed type II IGF receptor mRNA. Eleven of 11 patient
tumor specimens were positive (Fig. 3). The principal band in
this figure represents the protected fragment of the expected
size (377 base pairs). The less intense smaller bands appear to
be due to the presence of small quantities of riboprobe which
were synthesized at less than full template length, and so
represent an artifact of incomplete probe polymerization and
not separate RNA species. Of interest, the patient specimen
with the greatest amount of mRNA for the type II IGF receptor
in this study also contained the greatest amount of IGF-II
mRNA in our previously published work (6).
Insulin Receptor. Examination of breast tumor cell lines
showed all specimens (10 of 10) expressed mRNA for the
insulin receptor (Fig. 4).
IGF Binding Studies
IGF-I. IGF-I binding competition studies on MCF-7 cell
monolayers demonstrated a paradoxical two- to threefold in
crease in binding of label to the cells in the presence of small
quantities of unlabeled competing ligand (Fig. 5/1). This was
true for both unlabeled IGF-I and IGF-II. Similar biphasic
IGF-I binding results have been reported in previous studies of
human fibroblasts and have been interpreted to be due to the
interaction with IGF binding proteins which are released by the
cell during the binding assay (26, 27). Maximal IGF-I binding
to MCF-7 cells was seen in the presence of 1-2 pmol of
unlabeled IGF-I or 5-10 pmol of unlabeled IGF-II. With greater
concentrations of unlabeled competing ligand, labeled IGF-I
binding decreased. However, unlabeled IGF-I competed for this
binding with an apparent affinity that was approximately five
times greater than that of IGF-II. The results are consistent
with previous reports that IGF-I binds to the type I IGF receptor
with several-fold greater affinity than IGF-II (28-30). However,
some of the difference in competition seen could be accounted
for by binding of unlabeled IGF-II to the type II receptor.
Attempts to block binding of labeled IGF-I to the cells with
antibody to the type I IGF receptor (aIR-3) alone were difficult
to interpret since the amount of binding measured in the
absence of competing unlabeled ligand was only approximately
400 counts greater than the nonspecific binding. However, when
IGF-I binding was increased to the maximal level by the addi
tion of 5 ng unlabeled IGF-I, «IR-3demonstrated dose-depend
ent inhibition of binding (Fig. 6). At an antibody concentration
of 12 jug/ml, the concentration used to inhibit growth of MCF7 cells in a previous study (18), «IR-3blocked approximately
80% of specific IGF-I binding to the MCF-7 cell monolayer
(Fig. 6). Control mouse IgG did not inhibit IGF-I binding. The
ttIR-3 preparation used was purified by high-performance liquid
chromatography, and so would not be expected to contain any
-9.4
-5.2
-2.0
Fig. 2. Northern blot for type II IGF receptor mRNA in breast tumor cell
lines. Ten n% total RNA probed with a 377-base pair riboprobe for the type II
ICiF receptor. Hep G2. i hepatoccllular carcinoma line which overexpresses
mRNA for IGF-II is also included. A single 9.4-kilobase transcript is seen for all
specimens. Radiograph exposed 72 h.
// ////////{
f/
-527
'' •¿
CANCER
-404
-309
-242/238
<b .y
\" # oT^
y
j£ Ä£
/#
#
tomm
-527
-404
^"
-622
-242/238
-527
Fig. 3. RNasc protection assay for type II IGF receptor mRNA in cancer
epithelial cancer cell lines and breast tumor specimens. A. 30 jig total RNA from
tumor cell lines. All lines are from breast tumors except Hep G2 and SK Hep 1
(hepatocellular carcinomas). SK-OV3 (Ovarian carcinoma), SKNMC and CHP100 (neuroepitheliomas). SK-Ncp I (Wilm's tumor). 6401 (F.wing's sarcoma). B.
-404
20 /*g total RNA from patient tumor specimens. Fach set of initials represents a
different patient. RM and RM (node) represent tissue from a primary tumor and
a nodal metastasis in the same patient. Hep G2 and placenta! RNA included as
controls. A 377-base pair protected fragment is seen in all specimens. Radiographs
exposed 48 h.
Fig. 4. RNase protection assay for insulin receptor mRNA in breast cancer
cell lines. Thirty ^g total RNA from all cell lines. A 600-base pair protected
fragment is seen for all specimens. Radiograph exposed 48 h.
50
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1990 American Association for Cancer Research.
IGF RECEPTORS
IN BREAST
curve showed a K,, value for the type I receptor of 2 nM with 8
x 10' sites per cell.
IGF-II. Binding of labeled IGF-II to MCF-7 cells in monolayer culture is shown in Fig. 5. Scatchard analysis of this IGFII binding data shows a Kj, value of 1.6 nM with 1.0 x 10'
receptors/cell. While there is a very clear dose-dependent inhi
bition of binding with unlabeled IGF-II, there is little inhibition
of binding by unlabeled IGF-I. Additionally, «IR-3at concen
trations up to 20 Mg/ml did not have any effect on IGF-II
binding. Experiments with purified type I receptor have dem
onstrated that as an artifact of labeling, binding of this prepa
ration of'2'I-IGF-II to purified type I receptor is greatly reduced
(data not shown). This, combined with the approximately 10fold greater number of type II IGF receptors may explain why
unlabeled IGF-I had relatively little effect on labeled IGF-II
binding.
IGF-II binding competition studies showed that goat antiserum against the mannose 6-phosphate binding site of the type
II IGF receptor blocked approximately 75% of labeled IGF-II
binding to the cell monolayer at a titer of 1:40. However, this
was apparently due to the presence of binding protein in the
antiserum preparation, and subsequent experiments with affin
ity purified antibodies against the type II IGF receptor which
were able to block mannose 6-phosphate binding did not dem
onstrate significant inhibition of radiolabeled IGF-II binding
(data not shown).
A.
0
0.1
1.0
Concentration
10.0
of Cold Ligand (nM)
B.
y3000
^•„»^ •¿â€¢
—¿
"
-••—_i-^O-p-C.
.1''•••1:•
\^77-«^^o\*o^~~-o
25002000-150010005000
—¿
o°—
i
'3500
o-^o
'/I
10.1
Growth Studies
1
C•
1.0
.
Concentration
Treatment of MCF-7 cells with either 5 nM IGF-I or 10 n\i
IGF-II resulted in a three- to fourfold increase in cell number
after 4 days of growth compared with control cells grown in
serum free medium without IGF, consistent with previous
results (6). Treatment with 12 ng/m\ «IR-3blocked approxi
mately 80% of the stimulatory effect of both IGF-I and IGF-II
(Fig. 1A), whereas aIR-3 by itself did not significantly alter the
growth of MCF-7 cells in either serum free media or phenolred free IMEM with 10% fetal calf serum. Control mouse IgG
at a concentration of 12 ng/m\ had no stimulatory or inhibitory
effect on cell growth, and did not alter the mitogenic effects of
either IGF-I or IGF-II (Fig. IB).
Recombinant human insulin stimulated growth of MCF-7
cells in a dose-dependent fashion, (Fig. 8) however, «IR-3did
not block the mitogenic effects of insulin at any dose level.
10.0
.
of Cold Ligand (nM)
Fig. 5. Kadiulabcled ICil-' binding studies for MCF-7 cells in monolayer
culture. A. radiolabelcd IGF-I binding displacement study. 125I-IGF-I was incu
bated with confluent MCF-7 cells in the presence of increasing amounts of
unlabcled IGF-I (•)or IGF-II (O). B. radiolabeled IGF-II binding displacement
study. '"I-IGF-II was incubated with confluent MCF-7 cells in the presence of
increasing amounts of unlabcled IGF-1 (•)or IGF-II (O). All binding carried out
at 4'C for 4 h.
120 n
100Bl
60
:•
f
CANCER
40-
DISCUSSION
-> -
Growth assays presented in this and previous studies (6, 31)
demonstrate that both IGF-I and IGF-II can be potent mitogens
for human breast cancer cells in culture. We have shown that
IGF-I and IGF-II mRNA are easily detectable in the majority
of human breast cancer specimens (6, 8), and the pattern of
expression seen on in situ hybridization studies suggests one or
both of these factors may be acting in a paracrine fashion to
promote tumor growth (8). Since numerous studies have dem
onstrated significant cross reactivity between insulin, IGF-I,
IGF-II and their respective receptors, we performed a series of
experiments in an attempt to clarify the biological significance
of the interactions between the various ligands and receptors in
this system.
Expression of mRNA for the insulin receptor as well as the
type I and type II IGF receptors appears to be virtually universal
in the breast cancer cell lines and tumor specimens we exam
ined.
Cell growth experiments in MCF-7 cells show that blockade
0.010
•¿
0.100
1.000
10.000
Antibody Concentration
Fig. 6. Radiolabelcd 1GF-I binding study for MCF-7 cells in monohncr culture
in the presence of antibody against the type I IGF receptor. '"I-IGF-I »as
incubated with confluent MCF-7 cells in the presence of increasing amounts of
control mouse IgG (•)or aIR-.V an antibody against the type I IGF receptor (O).
contaminating IGF binding proteins, which could of themselves
inhibit IGF-I binding. A radioligand assay for the presence of
IGF binding protein demonstrated no binding protein activity
in the «IR-3preparation (data not shown).
Since the «IR-3experiment demonstrated that labeled IGFI binding to the cells in the presence of greater than 1 pmol of
unlabeled ligand was due to specific binding to the type I IGF
receptor, a Scatchard analysis of that portion of the binding
51
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1990 American Association for Cancer Research.
IGF RECEPTORS
IN BREAST
rh
y 200000
500000
400000t
\S
300000
200000
100000E-SFMSFU-HgG[1IGF-I
IGF-I
+alR3
IGF-I
+lgG
IGF-II
+ lgG
Fig. 7. Antibody inhibition of IGF mitogcncsis in MC'F-7 cells. All panels
represent cell number after 4 days of growth under the specified treatment
conditions. Starting cell concentration at day 0 was 25.000 cells/well. All counts
represent the mean of duplicate wells. Error barn, standard error of the mean. A.
cells grown in the presence (+) or absence (—)of 12 ng/ml i«IR-3.SFM, serum
free media: FCS. IMEM with 10^ fetal calf serum; IdF-l, SFM with 5 nM IGFI: IGF-II. SFM with 10 nM IGF-II. <ilR-3 inhibits most of the mitogenic effect
of both IGF-I and IGF-ll. B, control antibody treatment. Where indicated. IgG
(preimmune mouse IgG 12 »ig/ml)or «IR-3(12 Mg/ml) was added to the wells.
400000 •¿
300000
1•+1Insulin
" 200000
100000
04-SFM
0.1 /¿g
InsuÃœn
Ins in
fj.q
10//g
1.C-fi+nn•u
IGF-I
5nM
Fig. 8. aIR-3-mediated inhibition of insulin-mediated mitogenesis in MCF-7
cells. Cells were grown with specified concentrations of insulin in the presence
(+) or absence (-) of «IR-3.Counts represent the mean of duplicate wells after 4
days of growth under the conditions specified. Starting cell concentration was
25.000/well on day 0. Error ban. standard error of the mean. SFM. serum free
media; in.suiin. SFM plus insulin at O.I. 1.0, or 10 ^g/ml: KiF-l. SFM plus 5 n.M
IGF-I.
CANCER
logical effects of insulin in sensitive tissues are mediated via the
insulin receptor, numerous studies have shown that insulin can
bind with low affinity to the type I IGF receptor in cultured
breast tumor cells (29, 36). Previous studies in fibroblasts
suggested that the mitogenic effects of insulin, particularly at
high doses are mediated by the type I IGF receptor, because
insulin-stimulated thymidine uptake could be partially blocked
with «IR-3treatment (37). However, in the cell growth assay
employed here, «IR-3did not change the effect of insulin at
any dose level. These results are most consistent with the notion
that the mitogenic effect of insulin in this system does not
appear to rely on direct binding of insulin to the type I IGF
receptor, but is more likely the result of interaction directly
with the insulin receptor. Recent studies in cultured myoblasts
have suggested that phosphorylation of fi subunit of the type I
IGF receptor can result from insulin stimulated tyrosine kinase
activity of adjacent insulin receptors (38). If this mechanism is
correct, insulin-mediated phosphorylation of the type I IGF
receptor and subsequent intracellular events might not require
extracellular binding of insulin to the external domain of the
type I receptor, and so would not be blocked by «IR-3.
Since antibodies against the mannose-6 phosphate binding
site of the type II IGF receptor showed little blockade of IGFII binding in the radioligand assay, their absence of effect in
the cell growth assays is difficult to interpret. The lack of
inhibition of IGF-II binding by these antibodies does support
the observation noted in other studies that the binding domains
for mannose-6 phosphate and IGF-II on the type II IGF recep
tor are distinct (39).
The function of the type II IGF receptor in this system is
poorly understood. Its interaction with the mannose-6 phos
phate moiety of procathepsin D, or M, 52,000 protein, may be
important in the intracellular transport and packaging of this
proteolytic enzyme. This glycoprotein is produced in abundance
by a variety of breast tumors and cell lines, and is mitogenic for
some breast cancer epithelial cells (40, 41). More recently, it
has been suggested to activate the precursor form of transform
ing growth factor-«,another mitogen for breast cancer cells, by
cleaving the membrane bound precursor to the free mature
peptide (42).
Since available data suggest that both IGF-I and IGF-II may
be significant growth promoters in human breast cancer, the
evidence that the mitogenic effects of these hormones are
mediated by the type I IGF receptor will serve to narrow our
approach to biological manipulations which could interfere with
IGF effects and inhibit tumor growth.
REFERENCES
of the type I IGF receptor by «IR-3 blocked the mitogenic
effects of both exogenous IGF-I and IGF-II, but not insulin.
Since the radioligand studies confirmed that «IR-3does indeed
block specific binding of IGF-I to the type I receptor, but does
not block binding of IGF-II to the type II receptor, this argues
strongly that the mitogenic effects of both ligands are mediated
via the type I IGF receptor.
The receptor binding studies suggest that IGF-II binds to the
type I receptor 2-5 times less avidly than does IGF-I, and that
there is little binding of IGF-I to the type II IGF receptor. Both
of these findings are consistent with previous studies in the
literature (32, 33). This may explain in part the observation
that on a molar basis, IGF-I is a more potent mitogen than
IGF-II.
Insulin has been previously noted to be mitogenic for breast
tumor cells in culture (34, 35). While the predominant physio
1. Lippman. M. E.. Dickson. R. B.. Gelmann. E. P., Rosen, N., Knabbe, C..
Bates. S.. Bronzen. D., Huff. K.. and Kasid, A. Growth regulation of human
breast carcinoma occurs through regulated growth factor secretion. J. Cell.
Biochem.. 35: 1-16. 1987.
2. Blundell. T. L.. and Humbcl. R. V. Hormone families: pancreatic hormones
and homologous growth factors. Nature (Lond.). 287: 781-787, 1980.
3. Baxter. R. C. The somalomedins: insulin-like growth factors. Adv. Clin.
Chem., 25:49-115, 1986.
4. Nissley. S. P., and Rechler, M. M. Insulin-like growth factors: biosynthesis,
receptors, and carrier proteins. Hormonal Prot. Pept.. 12: 127-203, 1986.
5. Zapf. J.. and Froesch, V. R. Insulin-like growth factors/somatomedins:
structure, secretion, biological actions and physiological roles. Hormone
Res.. 24: 121-130. 1986.
6. Yee, D., Cullen. K. J.. Paik. S., Perdue, J. F.. Hampton. B., Schwartz, A.,
Lippman. M. E.. and Rosen, N. Insulin-like growth factor II mRNA expres
sion in human breast cancer. Cancer Res., 48: 6691-6696. 1988.
7. Karey. K. P., and Sirbasku, D. A. Differential responsiveness of human breast
cancer cell lines MCF-7 and T47-D to growth factors and 17-/Õestradiol.
Cancer Res., 48: 4083-4092. 1988.
8. Yee, D.. Paik, S., Lebovic, G.. Marcus, R.. Favoni. R.. Cullcn, K.. Lippman,
52
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1990 American Association for Cancer Research.
IGF RECEPTORS
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
IN BREAST CANCER
M., and Rosen, N. Analysis of IGF-I gene expression in malignancy—
evidence for a paracrine role in human breast cancer. Mol. Endocrinol., 3:
509-517, 1989.
Ullrich, A., Bell, J. R.. Chen. F. Y., Herrera, R„Petrozelli. M.. Dull, T. }..
Gray, A., et al. Human insulin receptor and its relation to the tyrosine kinase
family of oncogenes. Nature (Lond.), 313: 756-761, 1985.
Massague, J., and Czech, M. P. The subunit structures of two distinct
receptors for insulin like growth factors 1 and II and their relationship to the
insulin receptor. J. Biol. Chem., 257: 5038-5045, 1982.
Morgan, D. O., Edman, J. C., Standring. D. N.. Fried, V. A.. Smith. M. C.,
Roth. R. A., and Rutter. W. J. Insulin-like growth factor II receptor as a
multifunctional binding protein. Nature (Lond.), 329: 301-307, 1987.
Lobel, P., Dahms, N. M., Breitmeyer, J., Chirgwin, J. M., and Kornfeld, S.
Cloning of the bovine 215-kDa cation-independent mannose-six-phosphate
receptor. Proc. Nati. Acad. Sci. USA. 84: 2233-2237. 1987.
Rochcfort. H., Capony. F.. Cavalie-Barthez. G.. Chambón, M., Garcia, M..
Massot, O., Morisset, M., Touitou, 1., Vignon, F., and Westley, B. Estrogenregulated proteins and autocrine control of cell growth in breast cancer. Dev.
Oncol.. 43: 57-68, 1986.
Fradkin. J. E., Eastman, R. C., Lesniak, M. A., and Roth, J. Specificity
spillover at the hormone receptor-exploring its role in human disease. New
Engl. J. Med.. 320: 640-645, 1989.
Kiess, W., Haskell, J. F., Lee, L., Greenstein, L. A., Miller. B. E., Aarons,
A. L., Rechler, M. M., and Nissley, S. P. An antibody that blocks insulinlike growth factor (IGF) binding to the type II IGF receptor is neither an
agonist nor an inhibitor of IGF-stimulated biologic responses in L6 myoblasts. J. Biol. Chem., 262: 12745-12751, 1987.
Roth, R. A. Structure of the receptor for insulin-like growth factor II: the
puzzle amplified. Science (Wash. DC), 239: 1269-1271, 1988.
Artega, C. L., Kitten, L., Coronado, E., Jacobs, S., Kuli. F.. and Osborne, C.
K. Blockade of the type I somatomedin receptor inhibits growth of estrogen
receptor negative human breast cancer cells in athymic mice. (Abstract 683).
Proc. Ann. Meet. Endo. Soc., 1988.
Rohlik. Q. T., Adams, B.. Kull, F. C., and Jacobs, S. An antibody to the
receptor for insulin-like growth factor I inhibits the growth of MCF-7 cells
in tissue culture. Biochem. Biophys. Res. Commun., 149: 276-281, 1987.
Engel. L. W.. Young. N. A., Tralka. T. S., Lippman. M. E.. O'Brien, S. J.,
cells. J. Biol. Chem., 263: 2553-2563, 1988.
26. DcVroede, M. A., Tseng. L. Y., Katsoyannis. P. G.. Nissley, S. P., and
Rechler, M. M. Modulation of insulin-like growth factor I binding to human
fibroblast monolayer cultures by insulinlike growth factor carrier proteins
released to the incubation media. J. Clin. Invest., 77: 602-613, 1986.
27. Clemmons. D. R., Elgin, R. G.. Han. V. K. M.. Casella. S. J.. D'Ercole, A.
28.
29.
30.
31.
32.
33.
34.
35.
36.
and Joyce, M. J. Establishment and characterization of three new continuous
cell lines derived from human breast carcinoma. Cancer Res., 38: 33523364, 1978.
Darbre, P., Yates, J., Curtis, S., and King, R. J. B. Effects of estradiol and
tamoxifen on human breast cancer cells in serum-free culture. Cancer Res..
«.•349-354.1983.
Gowan, L. K., Hampton, B.. Hill, D. J., Schiuder. R. H.. and Perdue. J. F.
Purification and characterization of a unique high molecular weight form of
insulin-like growth factor II. Endocrinology. 121:449-458. 1987.
Nolan, C. M., Creek, K. E., Grubb, J. H., and Sly, W. S. Antibody to the
phosphomannosyl receptor inhibits recycling of the receptor in fibroblasts.
J. Cell Biochem., 35: 137-151, 1987.
Chirgwin. J. M., Przybyla, A. E., MacDonald. R. J., and Rutter, W. J.
isolation of biologically active ribonucleic acid from sources enriched in
ribonucleases. Biochemistry, /«:5294-5299, 1979.
Ullrich, A.. Gray, A.. Tam, A. W., Yang-Feng, T., Tsubokawa, M., Collins.
C., Henzel, W., LeBon. T., Kathuria, S., et al. Insulin-like growth factor I
receptor primary structure: comparison with insulin receptor suggests struc
tural determinants that define functional specificity. EMBO J.. 5: 25032512. 1986.
Oshima, A., Nolan. C. M.. Kule, J. W., Grubb, J. H., and Sly, W. S. The
human cation-independent mannose 6-phosphate receptor: cloning and se
quence of the full length cDNA and expression of functional receptor in COS
37.
38.
39.
40.
41.
42.
J.. and Van Wyk, J. J. Cultured fibroblast monolayers secrete a protein that
alters the cellular binding of somatomedin-c/insulin-like growth factor 1. J.
Clin. Invest., 77: 1548-1556, 1986.
Nissley, S. P., and Rechler. M. M. Somatomedin/insulin-like growth factor
tissue receptors. J. Clin. Endocrinol. Metab.. 13: 43-67. 1984.
Pollack, M. N.. Polychronakos. C., Yousefi. S.. and Richard. M. Character
ization of insulin-like growth factor I (IGF-I) receptors of human breast
cancer cells. Biochem. Biophys. Res. Commun., 154: 326-331. 1988.
Pekonen. F., Partanen. S., Makinen, T.. and Rutanen, E. M. Receptors for
epidermal growth factor and insulin-like growth factor I and their relation to
steroid receptors in human breast cancer. Cancer Res.. 48: 1343-1347, 1988.
Huff. K. K.. Kaufman. D., Gabbay, K. J.. Spencer. E. M., Lippman. M. E..
and Dickson. R. B. Human breast cancer cells secrete an insulin-like growth
factor-I related polypeptide. Cancer Res., 46: 4613-4619, 1986.
Ewton, D. Z., Falen, S. L., and Florini, J. R. The type II insulin-like growth
factor (IGF) receptor has low affinity for IGF-I analogs: pleiotypic actions
of IGFs on myoblasts are apparently mediated by the type I receptor.
Endocrinology. 120: 115-123. 1987.
Rosenfeld, R. G., Conover, C. A., Hodges, D., Lee, P. D., Misra, P.. Hint/.
R. L., and Li, C. H. Heterogeneity of insulin-like growth factor-I affinity for
the insulin-like growth factor-II receptor: comparison of natural, synthetic
and recombinant DNA-derived insulin-like growth factor I. Biochem. Bio
phys. Res. Commun.. 143: 199-205. 1987.
Osborne, C. K., Bolán.G., Monaco. M. E., and Lippman. M. li. Hormone
responsive human breast cancer in long-term culture: effect of insulin. Proc.
Nati. Acad. Sci. USA, 7.?:4536-4540. 1976.
Skarda, J., Green, C. D., Aisbitt. R. P. G.. and Barry, J. M. Insulin does not
stimulate DNA synthesis in cultured mammary tissue by enhancing glucose
uptake. J. Endocrinol.. 60: 197-198, 1974.
Furlanetto. R. W., and DiCarlo. J. N. Somatomedin-C receptors and growth
effects in human breast cells maintained in long-term tissue culture. Cancer
Res., 44:2122-2128, 1984.
Flier, J. S., Usher, P.. and Moses, A. C. Monoclonal antibody to the type I
insulin-like growth factor (IGF-I) blocks receptor-mediated DNA synthesis
clarification of the mitogenic mechanisms of K ,1 I and insulin in human
skin fibroblasts. Proc. Nail. Acad. Sci. USA, 83: 664-668. 1986.
Beguinot. F.. Smith, R. J.. Kahn. C. R.. Marón,R., Moses, A. C, and While,
M. F. Phosphorylation of Insulin-like growth factor I receptor by insulin
receptor tyrosine kinase in intact cultured skeletal muscle cells. Biochemistry,
27:3222-3228, 1988.
Braulke. T., Causin. C., Waheed, A., Junghans, U.. Hasilik, A.. Maly. P.,
Humbel, R. E., and von Figura. K. Mannose 6-phosphate/insulin-like growth
factor II receptor: distinct binding sites for mannose 6-phosphate and insulinlike growth factor II. Biochem. Biophys. Res. Commun.. ISO: 1287-1293,
1988.
Veith, F. O., Capony, F., Garcia. M.. Chantelard. J.. Pujol. H., Veith, F..
Zajvela. A., and Rochefort, H. Release of estrogen induced glycoprotein with
a molecular weight of 52,000 by breast cancer cells in primary culture. Cancer
Res.. 43: 1861-1868. 1983.
Vignon, F., Capony, F., Chambón, M., Press, G., Garcia. M., and Rochelort.
H. Autocrine growth stimulation of the MCF-7 breast cancer cells by the
estrogen-regulated 52K protein. Endocrinology. 118: 1537-1545. 1986.
Henry. J. L.. and Schultz. G. S. Release of transforming growth factor-a
from a nonsecreting human breast cell line by cathepsin-D. J. Cell. Biochem..
I3B: El 18. 1989.
53
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1990 American Association for Cancer Research.
Insulin-like Growth Factor Receptor Expression and Function in
Human Breast Cancer
Kevin J. Cullen, Douglas Yee, William S. Sly, et al.
Cancer Res 1990;50:48-53.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/50/1/48
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1990 American Association for Cancer Research.